The directive of the Human Genome Initiative is to determine the primary sequence of the intact human genome. The potential for matrix-assisted laser desorption/ionization mass spectrometry to play a significant role in this undertaking is dependent on the ability to generate stable gas-phase ions from large oligodeoxynucleotides that can be measured with sufficient sensitivity, resolution, and accuracy for their identification. The proposed research addresses current shortcomings in capabilities for generating stable gas-phase ions that can be measured accurately. Several major innovations will be developed during this research period. The problem of sensitivity will be addressed through analysis of backbone modified oligonucleotides. Chain extension utilizing alphaS-dNTPs will result in phosphorothioate oligonucleotides that can be sequenced via several protocols including a unique modified chemical alkylation procedure. The problem of oligonucleotide fragmentation will be solved by the addition of co-matrices to the MALDI experiment that reduce nucleobase protonation. The problem of mass resolution will be solved by the development of an improved hybrid TOF mass spectrometer. With the proposed methodology, DNA sequence information will be obtained from oligonucleotides up to the 400-mer level with fmol sensitivity and high mass accuracy and resolution. Further, these methods will be applicable to automation which will reduce the cost of sequencing efforts and improve the throughput of the number of bases that can be sequenced per day.